The present invention relates generally to methods and materials for quantitative detection of isoenzymes of creatine kinase having a B-subunit by radioimmunoassay techniques.
Creatine kinase is a dimeric molecule that exists in at least three isoenzyme combinations designated MM, MB and BB on the basis of monomer composition, M being the predominant form in muscle and B being the predominant form in brain. Plasma from normal human subjects contains primarily MM, with less than 0.005 international unit (IU) of MB per milliliter and no detectable BB CK. The only human tissue containing appreciable amounts of MB CK is myocardium so that elevated MB CK activity in plasma is a remarkably sensitive and specific marker indicator of myocardial injury. Similarly, after cerebral infarction, injury or infection, BB CK is released into the blood but only minor amounts appear in the circulation. This may be due to the blood brain barrier or to other factors. Since BB CK does not appear in human plasma after myocardial infarction and MB CK does not appear in brain after cerebral injury, the availability of a sensitive assay for the B-subunit should be useful in detecting and quantifying a myocardial infarction by measuring MB CK in plasma and detecting and quantifying the extent of cerebral injury after brain damage by measuring BB CK.
In the past, MB CK has been assayed in terms of its catalytic activity. Employing a spectrophotometric technique for detecting NADPH developed from creatine kinase--catalyzed formation of ATP through coupled enzyme systems, CK activity is measured before and after an antibody which will bind with MB CK is conjugated with the sample. The results of such prior art assays are directly dependent on several parameters and because of them, in general, enzyme immunoassays have lower sensitivity than radioimmunoassays. For one thing, the MB CK may be variably enzymatically inhibited by the antibody. For another, there is difficulty in distinguishing MM from MB activity because of the high background of MM CK activity inasmuch as MB CK never makes up more than 10 to 15 percent of total CK activity in plasma, even after acute myocardial infarction. The results also depend on the rate of disappearance of MB activity from plasma. Unfortunately, the factors responsible for disappearance of MB activity from the circulation have not been well elucidated. It is unclear, for example, whether disappearance of enzyme activity is rate limited by inactivation, denaturation or by removal of intact enzyme molecules from circulation. Similar problems are encountered in assaying BB CK by enzyme immunoassay.
The prior art provides certain proposals for radioimmunoassay ("RIA") analysis of plasma samples for use in quantitative detection of enzymes, and specifically creatine kinase isoenzymes, independently of analysis of enzyme activity. See, generally, the review article, "The Measurement of Enzymes by Radioimmunoassay" By J. Landon, et al. [Ann. Clin. Biochem., 14, pp. 90-99 (1977)] which recounts the relative superiority of RIA techniques to those based on catalytic activity. Simply put, according to one such RIA technique a stoichiometric excess of a pure, labelled (radioisotopic) material is allowed to associate (e.g., by antigen/antibody reaction) with the selected reactive substance such as antibody previously exposed to a sample containing an "unknown" quantity of enzyme which is unlabelled, but which is capable of a similar association. Direct quantitative information concerning the "unknown" concentration, as opposed to measuring enzymatic activity, is obtained on the basis of a count of radioactivity of the remaining selected substance which associates with the labelled material.
Nicholson et al., [Proc. Austral. Assoc. Neurologists, Vol. 10, pp. 105-108 (1973)] described a method involving labelling skeletal muscle MM creatine kinase with .sup.125 I and reported development of an RIA for the MM isoenzyme which assertedly measures "enzymes independently of the integrity of the active site." The method employed by Nicholson, et al. for labelling human creatine kinase is that described by Hunter, et al. [Nature, Vol. 194, pp. 4956 (1962)], employing chloramine-T to directly introduce the desired isotope into tyrosyl and histidyl residues of the enzyme protein chain. This method, however, has been associated with considerable structural changes (i.e., tertiary structure destruction) and loss of MM isoenzyme activity through disruption of the sulfhydryl-group-containing active site. Because the assay is assertedly specific for the M-subunit, it invites cross-reaction with MM CK and MB CK and is thus incapable of distinguishing elevation of serum MB concentration resulting from myocardial infarction from elevation of serum MM concentration resulting from skeletal muscle damage or disease. The chloramine-T reagent used in the labelling procedure of Nicholson, et al. cannot be used for labelling the BB or MB isoenzymes due to their relative lability (instability, vis-a-vis MM) in the presence of highly oxidative sulfhydryl-group-disrupting, reagents as chloramine-T. If the sulfhydryl group is disrupted, labelled antigen may not have quite the same affinity for the MB antiserum as unlabelled antigen. The consequence of this may show up in loss of sensitivity. In sum, the Nicholson, et al. proposal has not provided a useful basis to develop an RIA for BB and MB isoenzymes.
A prior art publication of interest to the background of the invention is Fang, et al. [Biochem. Biophys. Res. Comm., Vol. 65, pp. 413-419 (1975)] which reports that creatine kinase enzyme activity losses from direct iodination by prior art chloramine-T, thallic trichloride and lactoperoxidase methods may be avoided through use, for iodination, of a Bolton-Hunter acylation reagent which conjugates (combines) at a free amino (NH.sub.2) group of the protein, thus avoiding disruption of the active site. The reagent involved was an iodinated compound derived from N-succinimidyl-3-(4-hydroxyphenyl propionate). Fang, et al. labelled rabbit skeletal muscle MM CK. There is no mention of human MM CK nor was there any mention of animal or human MB or BB CK isoenzymes. While the reported preservation of enzyme active sites would have suggested that a more accurate RIA for MM creatine kinase than that of Nicholson, et al. might be secured, the prior art was still without a method for labelling the more labile MB and BB CK isoenzymes. Furthermore, the antibody noted in Fang, et al. was to rabbit MM CK which offered no specificity for human BB or MB CK isoenzymes. The necessary ingredients for a human CK isoenzyme RIA with a specific antibody to BB and MB CK and the necessary stabilizing conditions for such analysis were yet to be developed.
The radioimmunoassay described and claimed in the aforementioned application Ser. No. 846,095 was performed with antiserum to BB CK. In this assay, .sup.125 I-BB CK binding to BB antiserum was inhibited by unlabelled MB CK and formed the basis for measuring unknown amounts of MB CK in plasma. The specificity of MB CK antiserum for the B-subunit was also disclosed. While it would have been preferred to use .sup.125 I-MB CK and MB CK antiserum in a radioimmunoassay for MB CK, this was not feasible at that time since there was no method for complete purification of MB CK. The chief contaminant in MB CK is albumin which has a similar isoelectric point to MB CK and co-precipitates with it. Whereas impure MB CK could be used to displace binding of .sup.125 I-BB to BB antiserum, impure MB CK labelled with .sup.125 I could not be used since counts would also be on the albumin and would not be displaced with MB CK.
In the present application, a method for the purification of MB CK is disclosed. Purified MB CK is then radioisotopically labelled and a radioimmunoassay described and claimed with antiserum to MB CK. This assay has several advantages over the assay based on BB antiserum. MB CK is more stable than BB CK when labelled and has a longer shelf life making it more appropriate for routine clinical analysis. The assay also has greater reproducibility in that MB binding to MB antiserum is being displaced with MB rather than displacing BB binding to BB antiserum with MB CK. However, if purified MB is used with the BB system as described and claimed in Ser. No. 846,095, sensitivity and reproducibility are still adequate for diagnostic purposes.